The diagnostic evaluation and management of febrile infants, especially those 60 days of age and younger, remains a challenge for clinicians, especially in the emergency department (ED); Although most young febrile infants will have a non-bacterial infection, nearly 10% will have a serious bacterial infection (SBI), yet febrile infants with and without SBIs are often clinically indistinguishable. The currently recommended approach for the evaluation of febrile infants is inadequate and controversial as many of the routine screening tests for SBI are inaccurate. In the current era, however, there are novel methods, including advances in genomic sequencing and techniques for conducting high through-put deoxynucleic acid (DNA) and ribonucleic acid (RNA) analysis that have led to a better understanding of the host-pathogen response to infections. Thus, a novel approach to distinguish febrile infants infected with bacterial pathogens from those infected with non- bacterial pathogens is to examine the host response to infection. Recent data indicate that different pathogens induce distinct transcriptional biosignatures in the RNA of blood leukocytes that can be reliably measured by microarray analysis. We hypothesize that application of diagnostic biosignatures will allow accurate discrimination between young febrile infants evaluated in the ED with bacterial infections and those infected with non-bacterial pathogens. The eventual paradigm shift of how we evaluate young febrile infants will result in improved and less invasive diagnostic evaluations of febrile infants, and will have a significant and beneficial impact by reducing the number of (1) unnecessary invasive tests (including lumbar punctures), (2) use of empirical antibiotics, (3) hospitalizations and associated iatrogenic complications, and (4) emotional and financial burdens on families. In this prospective study, eligible febrile infants 60 days of age or younger who present to 22 participating EDs of the Pediatric Emergency Care Applied Research Network (PECARN) will be enrolled after informed consent is obtained. We will define and validate bacterial and non-bacterial diagnostic biosignatures from small volumes of blood obtained during routine clinical evaluation for SBI. Additionally, we will evaluate the newer screening test (procalcitonin) and compare its tests characteristics with the traditional screening tests, (complete blood counts) with the reference standard (microbiologic cultures of relevant specimens such as blood, CSF and urine) and the diagnostic biosignatures in the evaluation of febrile infants for SBI. PUBLIC HEALTH RELEVANCE: Fever is a very common reason for visits to the emergency department, and febrile infants younger than 60 days of age are at high risk for serious bacterial infections. Since the routinely used screening tests and clinical evaluations to identify these serious infections are not adequate, we are proposing the use of a novel diagnostic blood test which looks at the infant's response to the infection. This will allows us to reliably distinguish those with and without serious bacterial infections, which in turn will reduce; (a) a substantial number of unnecessary invasive tests, (b) use of unnecessary antibiotics, and (c) hospitalizations in young infants, thus, reducing the emotional burden on the families and costs to the U.S. health care system.